{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002114","ANALYSIS_ID":"AN003461","VERSION":"1","CREATED_ON":"March 21, 2022, 9:55 am"},

"PROJECT":{"PROJECT_TITLE":"Mass Spectrometric Data of Cardiolipin in Cells under Mieap Expression","PROJECT_SUMMARY":"Mass spectrometric data of cardiolipin in A549 (Ad-Mieap infected vs. non-infected) and LS174T cells (LS174T-cont vs. Mieap-KD)","INSTITUTE":"National Cancer Center Japan Research Institute","LAST_NAME":"Ikari","FIRST_NAME":"Naoki","ADDRESS":"5-1-1 Tsukiji, Chuo-ku, Chuo-ku, Tokyo, 104-0045, Japan","EMAIL":"nikari@ncc.go.jp","PHONE":"+81-3-3542-2511"},

"STUDY":{"STUDY_TITLE":"Involvement of Mieap in Cardiolipin metabolism (part I-revised)","STUDY_SUMMARY":"Cardiolipin (CL) alterations cause mitochondrial dysfunction. Mieap is involved in mitochondrial quality control (MQC). To investigate whether Mieap functions in MQC via regulation of CL metabolism, quantitative assessment of total CL and comparison of CL species conducted with A549 (Ad-Mieap infected vs. non-infected) and LS174T cells (LS174T-cont vs. Mieap-KD). The A549 cells were harvested 24 hr after infection with Ad-Mieap and were compared with non-infected cells by mass spectrometric analysis. The LS174T-cont and Mieap-KD cells incubated under a normal condition were harvested and subjected to mass spectrometric analysis.","INSTITUTE":"National Cancer Center Japan Research Institute","LAST_NAME":"Ikari","FIRST_NAME":"Naoki","ADDRESS":"5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan","EMAIL":"nikari@ncc.go.jp","PHONE":"+81335422511"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_BIOSOURCE_OR_SUPPLIER":"American Type Culture Collection","CELL_STRAIN_DETAILS":"A549 (tissue, lung cancer; gender, male), LS174T (tissue, colon cancer; gender, female)","CELL_COUNTS":"10,000,000"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"A549 Ad-Mieap 1",
"Factors":{"Genotype/Treatment":"Ad-Mieap infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Non-infection 1",
"Factors":{"Genotype/Treatment":"Non-infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Ad-Mieap 2",
"Factors":{"Genotype/Treatment":"Ad-Mieap infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Non-infection 2",
"Factors":{"Genotype/Treatment":"Non-infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Ad-Mieap 3",
"Factors":{"Genotype/Treatment":"Ad-Mieap infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Non-infection 3",
"Factors":{"Genotype/Treatment":"Non-infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Ad-Mieap 4",
"Factors":{"Genotype/Treatment":"Ad-Mieap infection"}
},
{
"Subject ID":"-",
"Sample ID":"A549 Non-infection 4",
"Factors":{"Genotype/Treatment":"Non-infection"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 1",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 1",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 2",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 2",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 3",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 3",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 4",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 4",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 5",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 5",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Control 6",
"Factors":{"Genotype/Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"LS174T Mieap-KD 6",
"Factors":{"Genotype/Treatment":"Mieap-knockdown"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were collected using trypsin-EDTA. The cells were snap-frozen in liquid nitrogen after cell count, and subsequently stored at -80°C until lipidomic analysis.","SAMPLE_TYPE":"Cultured cells","VOLUMEORAMOUNT_COLLECTED":"10,000,000 cells/tube","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Infection of the A549 cell line was carried out by adding viral solution (Ad-Mieap) to A549 cell monolayers, and incubating at 37°C for 120 min with brief agitation every 20 min. This was followed by the addition of culture medium and the return of the infected cells to the 37°C incubator. We established a Mieap-KD cell line using LS174T. Mieap expression was inhibited in the cell line by retroviral expression of short-hairpin RNA (shRNA) against the Mieap sequence. We also established LS174T-cont cells using the retroviral vector with target sequence for EGFP. The LS174T-cont and Mieap-KD cells were incubated under normal condition.","TREATMENT_DOSEDURATION":"A549 cells: 24 h; LS174T-cont and Mieap-KD cells: none (incubated under normal condition)","TREATMENT_VEHICLE":"A549 cells: viral solution (Ad-Mieap); LS174T-cont and Mieap-KD cells: none (incubated under normal condition)","CELL_STORAGE":"stored at -80°C"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Total lipids were extracted from samples using the Bligh-Dyer method. An aliquot of the organic phase was added to an equal volume of methanol before being loaded onto a DEAE-cellulose column (Wako Chemical) pre-equilibrated with chloroform. After successive washes with chloroform/methanol (1:1, v/v), acidic phospholipids were eluted with chloroform/methanol/HCl/water (12:12:1:1, v/v), followed by evaporation to dryness to yield a residue was soluble in methanol.","EXTRACTION_METHOD":"the Bligh-Dyer method"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"UltiMate 3000 (Thermo Fisher Scientific)","COLUMN_NAME":"X-Bridge C18 column (3.5 μm, 150 mm × 1.0 mm i.d.) (Waters)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"pmol/10,000,000 cells"},

"MS_METABOLITE_DATA":{
"Units":"pmol/10E6 cells",

"Data":[{"Metabolite":"CL(14:0_16:1_18:1_14:0)","A549 Ad-Mieap 1":"9.81","A549 Non-infection 1":"6.63","A549 Ad-Mieap 2":"9.43","A549 Non-infection 2":"8.86","A549 Ad-Mieap 3":"11.32","A549 Non-infection 3":"7.03","A549 Ad-Mieap 4":"8.86","A549 Non-infection 4":"8.31","LS174T Control 1":"7.07","LS174T Mieap-KD 1":"5.64","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"8.31","LS174T Mieap-KD 3":"5.90","LS174T Control 4":"7.68","LS174T Mieap-KD 4":"6.45","LS174T Control 5":"10.42","LS174T Mieap-KD 5":"7.03","LS174T Control 6":"5.55","LS174T Mieap-KD 6":"5.95"},{"Metabolite":"CL(15:0_16:1_15:0_16:1)","A549 Ad-Mieap 1":"0.00","A549 Non-infection 1":"0.00","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"0.00","LS174T Mieap-KD 1":"0.00","LS174T Control 2":"8.42","LS174T Mieap-KD 2":"7.54","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(14:0_16:1_14:0_18:2)","A549 Ad-Mieap 1":"18.04","A549 Non-infection 1":"11.54","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"43.29","LS174T Mieap-KD 1":"48.82","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(16:1_16:1_16:1_14:0)","A549 Ad-Mieap 1":"0.46","A549 Non-infection 1":"0.15","A549 Ad-Mieap 2":"20.03","A549 Non-infection 2":"17.81","A549 Ad-Mieap 3":"20.12","A549 Non-infection 3":"10.60","A549 Ad-Mieap 4":"18.09","A549 Non-infection 4":"18.03","LS174T Control 1":"4.35","LS174T Mieap-KD 1":"4.24","LS174T Control 2":"29.51","LS174T Mieap-KD 2":"22.29","LS174T Control 3":"46.03","LS174T Mieap-KD 3":"47.07","LS174T Control 4":"46.21","LS174T Mieap-KD 4":"59.15","LS174T Control 5":"74.23","LS174T Mieap-KD 5":"58.40","LS174T Control 6":"48.13","LS174T Mieap-KD 6":"54.65"},{"Metabolite":"CL(16:1_14:1_16:1_16:1)","A549 Ad-Mieap 1":"2.01","A549 Non-infection 1":"1.15","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"10.40","LS174T Mieap-KD 1":"14.81","LS174T Control 2":"7.15","LS174T Mieap-KD 2":"7.17","LS174T Control 3":"9.95","LS174T Mieap-KD 3":"15.14","LS174T Control 4":"10.31","LS174T Mieap-KD 4":"19.57","LS174T Control 5":"14.15","LS174T Mieap-KD 5":"15.75","LS174T Control 6":"10.20","LS174T Mieap-KD 6":"14.78"},{"Metabolite":"CL(16:1_16:1_12:0_18:2)","A549 Ad-Mieap 1":"0.00","A549 Non-infection 1":"0.00","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"2.07","A549 Non-infection 3":"0.98","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"0.00","LS174T Mieap-KD 1":"0.00","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(16:0_16:0_16:0_16:0)","A549 Ad-Mieap 1":"21.12","A549 Non-infection 1":"0.01","A549 Ad-Mieap 2":"8.99","A549 Non-infection 2":"0.58","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.11","A549 Non-infection 4":"1.64","LS174T Control 1":"0.00","LS174T Mieap-KD 1":"0.00","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.02","LS174T Mieap-KD 3":"65.64","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(14:0_16:0_16:0_18:1)","A549 Ad-Mieap 1":"1.31","A549 Non-infection 1":"3.39","A549 Ad-Mieap 2":"1.68","A549 Non-infection 2":"6.40","A549 Ad-Mieap 3":"1.67","A549 Non-infection 3":"3.71","A549 Ad-Mieap 4":"1.51","A549 Non-infection 4":"6.79","LS174T Control 1":"0.68","LS174T Mieap-KD 1":"0.40","LS174T Control 2":"0.86","LS174T Mieap-KD 2":"0.38","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(16:0_16:1_14:0_18:1)","A549 Ad-Mieap 1":"17.73","A549 Non-infection 1":"14.80","A549 Ad-Mieap 2":"13.70","A549 Non-infection 2":"17.50","A549 Ad-Mieap 3":"20.92","A549 Non-infection 3":"15.47","A549 Ad-Mieap 4":"13.10","A549 Non-infection 4":"18.18","LS174T Control 1":"6.39","LS174T Mieap-KD 1":"4.92","LS174T Control 2":"5.21","LS174T Mieap-KD 2":"3.74","LS174T Control 3":"7.92","LS174T Mieap-KD 3":"5.64","LS174T Control 4":"7.78","LS174T Mieap-KD 4":"6.25","LS174T Control 5":"9.14","LS174T Mieap-KD 5":"6.16","LS174T Control 6":"5.42","LS174T Mieap-KD 6":"5.00"},{"Metabolite":"CL(14:0_16:1_18:1_16:1)","A549 Ad-Mieap 1":"99.45","A549 Non-infection 1":"68.56","A549 Ad-Mieap 2":"92.90","A549 Non-infection 2":"74.27","A549 Ad-Mieap 3":"113.68","A549 Non-infection 3":"67.00","A549 Ad-Mieap 4":"87.62","A549 Non-infection 4":"72.66","LS174T Control 1":"93.27","LS174T Mieap-KD 1":"79.70","LS174T Control 2":"73.33","LS174T Mieap-KD 2":"49.13","LS174T Control 3":"107.84","LS174T Mieap-KD 3":"81.50","LS174T Control 4":"121.80","LS174T Mieap-KD 4":"102.33","LS174T Control 5":"151.02","LS174T Mieap-KD 5":"97.73","LS174T Control 6":"79.92","LS174T Mieap-KD 6":"85.37"},{"Metabolite":"CL(16:1_16:1_16:1_16:1)","A549 Ad-Mieap 1":"73.98","A549 Non-infection 1":"51.06","A549 Ad-Mieap 2":"89.07","A549 Non-infection 2":"75.37","A549 Ad-Mieap 3":"85.76","A549 Non-infection 3":"46.96","A549 Ad-Mieap 4":"81.06","A549 Non-infection 4":"75.21","LS174T Control 1":"207.64","LS174T Mieap-KD 1":"228.20","LS174T Control 2":"187.63","LS174T Mieap-KD 2":"125.88","LS174T Control 3":"226.89","LS174T Mieap-KD 3":"228.01","LS174T Control 4":"263.03","LS174T Mieap-KD 4":"321.83","LS174T Control 5":"404.50","LS174T Mieap-KD 5":"300.64","LS174T Control 6":"254.03","LS174T Mieap-KD 6":"274.20"},{"Metabolite":"CL(18:2_14:1_16:1_16:1)","A549 Ad-Mieap 1":"4.81","A549 Non-infection 1":"2.95","A549 Ad-Mieap 2":"4.66","A549 Non-infection 2":"4.23","A549 Ad-Mieap 3":"5.09","A549 Non-infection 3":"2.72","A549 Ad-Mieap 4":"4.59","A549 Non-infection 4":"4.38","LS174T Control 1":"11.67","LS174T Mieap-KD 1":"16.65","LS174T Control 2":"8.16","LS174T Mieap-KD 2":"6.61","LS174T Control 3":"11.76","LS174T Mieap-KD 3":"16.52","LS174T Control 4":"12.21","LS174T Mieap-KD 4":"20.33","LS174T Control 5":"17.84","LS174T Mieap-KD 5":"17.41","LS174T Control 6":"11.86","LS174T Mieap-KD 6":"16.23"},{"Metabolite":"CL(14:2_18:2_16:1_16:1)","A549 Ad-Mieap 1":"0.01","A549 Non-infection 1":"0.00","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"0.21","LS174T Mieap-KD 1":"0.36","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(18:4_14:0_16:1_16:1)","A549 Ad-Mieap 1":"1.88","A549 Non-infection 1":"0.95","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"2.04","LS174T Mieap-KD 1":"2.51","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(16:0_16:0_18:0_16:0)","A549 Ad-Mieap 1":"3.72","A549 Non-infection 1":"0.20","A549 Ad-Mieap 2":"0.00","A549 Non-infection 2":"0.00","A549 Ad-Mieap 3":"0.00","A549 Non-infection 3":"0.00","A549 Ad-Mieap 4":"0.00","A549 Non-infection 4":"0.00","LS174T Control 1":"0.00","LS174T Mieap-KD 1":"0.00","LS174T Control 2":"0.00","LS174T Mieap-KD 2":"0.00","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.00","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"0.00","LS174T Mieap-KD 5":"0.00","LS174T Control 6":"0.00","LS174T Mieap-KD 6":"0.00"},{"Metabolite":"CL(16:0_16:0_16:0_18:1)","A549 Ad-Mieap 1":"3.17","A549 Non-infection 1":"7.72","A549 Ad-Mieap 2":"2.94","A549 Non-infection 2":"15.33","A549 Ad-Mieap 3":"3.51","A549 Non-infection 3":"7.36","A549 Ad-Mieap 4":"2.56","A549 Non-infection 4":"16.87","LS174T Control 1":"0.91","LS174T Mieap-KD 1":"0.43","LS174T Control 2":"0.62","LS174T Mieap-KD 2":"0.21","LS174T Control 3":"0.00","LS174T Mieap-KD 3":"0.00","LS174T Control 4":"0.70","LS174T Mieap-KD 4":"0.00","LS174T Control 5":"1.59","LS174T Mieap-KD 5":"0.58","LS174T Control 6":"1.59","LS174T Mieap-KD 6":"0.63"},{"Metabolite":"CL(14:0_18:1_16:0_18:1)","A549 Ad-Mieap 1":"47.68","A549 Non-infection 1":"44.62","A549 Ad-Mieap 2":"42.32","A549 Non-infection 2":"52.70","A549 Ad-Mieap 3":"59.92","A549 Non-infection 3":"49.96","A549 Ad-Mieap 4":"40.05","A549 Non-infection 4":"56.86","LS174T Control 1":"12.17","LS174T Mieap-KD 1":"8.21","LS174T Control 2":"11.40","LS174T Mieap-KD 2":"4.30","LS174T Control 3":"13.19","LS174T Mieap-KD 3":"7.59","LS174T Control 4":"18.28","LS174T Mieap-KD 4":"5.45","LS174T Control 5":"27.25","LS174T Mieap-KD 5":"10.48","LS174T Control 6":"15.96","LS174T Mieap-KD 6":"9.63"},{"Metabolite":"CL(14:0_18:1_16:1_18:1)","A549 Ad-Mieap 1":"160.59","A549 Non-infection 1":"127.72","A549 Ad-Mieap 2":"136.68","A549 Non-infection 2":"129.08","A549 Ad-Mieap 3":"225.56","A549 Non-infection 3":"146.76","A549 Ad-Mieap 4":"130.14","A549 Non-infection 4":"130.89","LS174T Control 1":"90.37","LS174T Mieap-KD 1":"68.64","LS174T Control 2":"78.79","LS174T Mieap-KD 2":"50.25","LS174T Control 3":"123.28","LS174T Mieap-KD 3":"77.65","LS174T Control 4":"133.95","LS174T Mieap-KD 4":"90.63","LS174T Control 5":"136.79","LS174T Mieap-KD 5":"88.85","LS174T Control 6":"76.56","LS174T Mieap-KD 6":"75.03"},{"Metabolite":"CL(18:2_14:0_16:1_18:1)","A549 Ad-Mieap 1":"374.87","A549 Non-infection 1":"287.41","A549 Ad-Mieap 2":"459.29","A549 Non-infection 2":"354.15","A549 Ad-Mieap 3":"511.04","A549 Non-infection 3":"308.17","A549 Ad-Mieap 4":"424.75","A549 Non-infection 4":"364.52","LS174T Control 1":"506.35","LS174T Mieap-KD 1":"445.20","LS174T Control 2":"582.08","LS174T 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}

}